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Mechanism of plasmid pT181 DNA replication
S A Khan1, R W Murray, R R Koepsel
1Department of Microbiology, Biochemistry and Molecular Biology, University of Pittsburgh, School of Medicine, PA 15261.
Biochimica Et Biophysica Acta
|December 20, 1988
Summary
Plasmid pT181 replication initiates when the RepC protein nicks the origin. This study reveals that the leading-strand origin also functions as the replication termination site, generating single-stranded DNA intermediates.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Plasmid pT181 replication is initiated by the RepC protein, which nicks the origin of replication.
- This nick is presumed to be the starting point for DNA replication via extension synthesis.
Purpose of the Study:
- To investigate the mechanism of pT181 replication, specifically the role of the origin sequence.
- To determine if the origin of replication also serves as a termination site.
- To elucidate the nature of DNA intermediates during pT181 replication.
Main Methods:
- In vitro replication assays using plasmid pT181.
- Construction and analysis of recombinant plasmids with two pT181 origins in direct and inverted orientations.
- Characterization of single-stranded DNA products generated during replication.
Main Results:
- In vitro replication produced both double-stranded and circular single-stranded DNA (leading strand).
- Recombinant plasmids with directly oriented origins showed initiation at one origin and termination at the other.
- Recombinant plasmids with inversely oriented origins initiated replication at one origin and stalled at the other.
- The pT181 leading-strand origin functions as both the initiation and termination site.
Conclusions:
- Plasmid pT181 replication proceeds via a rolling circle mechanism.
- Single-stranded DNA is a key intermediate in pT181 replication.
- The origin of replication is a dual-function site for initiation and termination.